Canine Mammary Gland Research Articles

Immunocytochemical detection of Ki‐67 in Diff‐Quik‐stained cytological smears of canine mammary gland tumours

To investigate whether Diff-Quik stained fine needle aspirate smears can be used to evaluate Ki-67 expression by immunocytochemistry. Both cytological and histological samples were obtained from 24 dogs with spontaneously developed mammary gland tumours. The cytological and histological specimens were examined by Diff-Quik and H&E stains, respectively. After examination, both samples were immunostained using the same Ki-67 antibody. The % Ki-67 values were calculated based on the percentage of positively stained tumour cells per 500 and 1000 tumour cells in cytology and histology specimens, respectively. Ki-67 staining was successful in 17/24 smears (71%) and 19/23 sections (83%). The correlation coefficient between the percentage of Ki-67-positive cells in cytological smears and in the histological sections was 0.677 (P < 0.01). These values were significantly different between histologically benign and malignant tumour groups both in cytology and histology samples (P < 0.001). The threshold value of the percentage of Ki-67-positive cells for distinguishing benign from malignant tumours was set at 4.85% with 90.9% sensitivity and 92.3% specificity by Receiver Operating Characteristic (ROC) curve using histopathology as the gold standard. Diff-Quik-stained cytology smears can be used to detect the presence of Ki-67 antigen when histology sections are not available.

Immunohistochemical evaluation of MMP-2 and TIMP-2 in canine mammary tumours: A survival study

Canine mammary tumours (CMTs) are a very heterogeneous group of neoplasms with variable prognosis. Their aggressiveness is mainly due to their ability to invade locally and to metastasize. The degradation of extracellular matrix components is an important determinant of the invasive phenotype. The aims of this study were to analyse by immunohistochemistry and double immunofluorescence the expression of metalloproteinase 2 (MMP-2) and tissue inhibitor of metalloproteinase 2 (TIMP-2) in eight normal canine mammary glands and 118 CMTs (24 benign, 94 malignant) and to investigate relationships with metastatic disease and survival. MMP-2 and TIMP-2 expression was higher in malignant tumours than in normal canine mammary tissue and benign tumours. The main difference between benign and malignant CMTs was the pattern of expression of both molecules: benign tumours presented TIMP-2 and MMP-2 immunoreactivity in the myoepithelial cells lining the basement membrane of tubuloalveolar structures, while malignant tumours showed mainly diffuse expression in neoplastic cells. In malignant tumours, increased TIMP-2 expression was significantly associated with the development of distant metastases, lower overall survival and lower disease-free survival. MMP-2 expression was not significantly associated to any of these parameters. These results suggest that the immunohistochemical expression of TIMP-2 is a useful prognostic factor in CMTs.

Expression of 14-3-3 σ protein in normal and neoplastic canine mammary gland

14-3-3 σ protein is a negative cell cycle regulator, with both reduced and elevated levels associated with cancer in humans. This study assessed the expression of this protein in canine mammary tissues using immunohistochemistry and Western blotting. 14-3-3 σ was detected in 97% of the mammary tissue samples examined and was found in both myoepithelial (MECs) and epithelial (ECs) cells. Expression levels were elevated and reduced in neoplastic ECs and MECs, respectively (P<0.001). Intense expression of 14-3-3 σ was detected in neoplastic ECs infiltrating blood vessels and lymph nodes and suggests a possible role for this protein in the malignant transformation of mammary neoplasms. Moreover, double immunostaining for 14-3-3 σ and the MEC-specific marker p63, confirmed that 14-3-3 σ is a highly sensitive marker of MECs since all p63-positive cells were also positive for 14-3-3 σ. However, this protein is not exclusive to MECs as ECs also labelled positively.

Effects of NF-.KAPPA.B Expression and Its Inhibition on Canine Mammary Cancer Cell Lines in an Immunodeficient Mice Model

Two canine mammary gland tumor (MGT) cell lines, CHMp-5b and -13a, were transplanted into nude mice, and their tumor development and metastatic potential were evaluated. In addition, NF-κB, Ki-67 and cyclin D1 expressions in the tumor masses were evaluated, and the relationship between these expressions and malignancy of the tumors developed in nude mice was investigated. Lower activation of NF-κB was positively correlated with a lower potential of metastasis and better prognosis in nude mice xenografted with CHMp-13a compared with CHMp-5b. Then, CHMp-5b cells were treated with BAY11-7082, an inhibitor of NF-κB, and the expressions of I-κBα, p-I-κBα, cyclin D1 and Bcl-2 in these cells were evaluated by Western blot analysis. In addition, BAY11-7082 at a dose of 6 mg/kg was administered intraperitoneally to nude mice xenografted with CHMp-5b, and relationships between tumor growth and lung and lymph node metastasis in nude mice and NF-κB, I-κBα and p-I-κBα expressions in tissues of these mice were evaluated. BAY11-7082 treatment induced decreased expressions of p-I-κBα, cyclin D1 and Bcl-2 in a dose- and time-dependent manner, suggesting that BAY inhibited NF-κB in this cell line. CHMp-5b-xenografted mice treated with BAY11-7082 showed a reduction in tumor growth and metastasis. Therefore, inhibition of the NF-κB signaling pathway may be a promising novel therapeutic approach for canine MGTs.

Substantial Interobserver Variation Among Morphopathological Diagnosis Of Canine Mammary Gland Tumours From Veterinary And Human Pathologists

Because the morphopathological diagnosis for the benignity or malignance of tumours has played a key role of therapeutic decisions and prognosis, the reproducibility and coherence among pathologists is important. To determine the level of interobserver variation for comparative studies among veterinary and human pathologists for the diagnosis of canine mammary gland tumours (MGTs), one hundred and thirty-six selected canine MGTs with histological slides were randomly divided into two groups: 68 cases were evaluated independently by three veterinary pathologists and one human pathologist; other 68 cases were reviewed blindly by one veterinary pathologists and three human pathologists. All the participating pathologists designated whether the canine MGTs are benign or malignant. Kappa (κ) statistics were calculated to evaluate the level of agreement. For three human pathologists involved in this study, moderate to good levels of agreement among their diagnosis (κ=0.68-0.81). However, three veterinary pathologists participated in this study show less than chance to slight levels agreement (κ= -0.06 to 0.25). In this study, human pathologists tend to diagnosed canine MGTs as benign lesions while veterinary pathologists tend to diagnose as malignant lesions. We conclude that substantially high interobserver variations do exist among pathologists and will greatly influence the research result and therefore; internationally accepted pathological diagnostic definitions with high accuracy and reproducibility among pathologists are required to assist global researchers in characterizing the tumour biology, natural history, and the comparison of treatment modalities of canine MGTs.

Characterization of spheres derived from canine mammary gland adenocarcinoma cell lines

There is increasing evidence for the presence of cancer stem cells in several solid tumors, and these cancer stem cells have a potential role in tumor initiation, aggression, and recurrence. The stem cell-like properties of spheres derived from canine mammary tumors remain largely elusive. We attempted to induce sphere formation using four cell lines of canine mammary adenocarcinoma, and characterized the spheres derived from a CHMp line in vitro and in vivo. The CHMp-derived spheres showed predominantly CD44 +CD24 − population, higher expression of stem cell-related genes, such as CD133, Notch3 and MDR, and higher resistance to doxorubicin compared with the CHMp-derived adherent cells. Xenograft transplantations in nude mice demonstrated that only 1 × 10 4 sphere cells were sufficient for tumor formation. Use of the sphere assay on these sphere-derived tumors showed that sphere-forming cells were present in the tumors, and were maintained in serial transplantation. We propose that spheres derived from canine mammary adenocarcinoma cell lines possess a potential characteristic of cancer stem cells. Spheres derived from canine mammary tumors could be a powerful tool with which to investigate novel therapeutic drugs and to elucidate the molecular and cellular mechanisms that underlie tumorigenesis.

Development, Anatomy, Histology, Lymphatic Drainage, Clinical Features, and Cell Differentiation Markers of Canine Mammary Gland Neoplasms

Mammary neoplasms are the most common neoplasm in female dogs. This article describes the embryologic development, normal anatomy, and histology of the canine mammary gland from the onset of first estrous and the changes that occur in the mammary gland during the estrus cycle. The clinical features of canine mammary gland tumors and their relation to prognosis are discussed, including age, hormones, breed, diet, and obesity. Additional clinical prognostic factors including clinical presentation, tumor size, and lymph node status at the time of presentation are discussed in relation to diagnosis and tumor staging. Immunohistochemical evaluation of the cell differentiation markers of the normal and neoplastic canine mammary gland is described and compared with similar studies in humans; the ways these markers may be used to assist with the prognosis of canine mammary neoplasms are discussed.

39 IN VITRO DEVELOPMENT OF CANINE EMBRYOS PRODUCED BY INTERSPECIES SOMATIC CELL NUCLEAR TRANSFER USING ENUCLEATED BOVINE OOCYTES

Interspecies somatic cell nuclear transfer (iSCNT) is an invaluable tool for studying nucleous-cytoplasm interactions, and may provide an alternative for cloning endangered animals, whose oocytes are difficult to obtain. Using readily available oocytes from domestic/farm animals as recipients for iSCNT would greatly benefit ongoing research on somatic cell reprogramming. However, little information is available concerning the development of canine iSCNT embryos reconstructed with bovine oocyte cytoplasm. In the first experiment, we investigated the influence of donor cell type on the development of canine iSCNT embryos reconstructed with enucleated bovine oocytes. Canine mammary gland tumour (MGT) cells and cumulus cells were used as donor cell. The bovine oocytes matured for 22 h were enucleated by the micromanipulator, and the donor cells were transferred into the perivitelline space adjacent to the plasma membrane of the oocyte. The couples were fused and activated simultaneously with a single DC pulse of 2.3 kV cm–1 for 30 μs, using an electro cell fusion generator. The reconstructed embryos were cultured for 72 h in the mSOF medium supplemented with 0.4% BSA. After 72 h of culture, only cleaved embryos were further co-cultured with bovine cumulus cells in mSOF supplemented with 5% fetal bovine serum (FBS) for an additional 5 days. In the second experiment, we examined the effects of serum type on the development of canine iSCNT embryos. The embryos reconstructed with canine cumulus cells were co-cultured with canine cumulus cells in mSOF supplemented with 5% FBS, and canine oestrous and diestrous serum for 5 days after 72 h of culture with 0.4% BSA. Data were analysed by chi-square analysis with a Yates’ correction. More than 75% of the canine somatic cells successfully were fused with bovine enucleated oocytes following electrofusion, irrespective of the types of the donor cells. There were no significant differences in the cleavage rates of iSCNT embryos between the cumulus cell and MGT cell (66.2% v. 62.6%). Although none of the embryos reconstructed with MGT cells (n = 123) developed to the 16-cell stage, 6% of embryos with cumulus cells (n = 133) reached at least the 16-cell stage. There were no significant differences in the cleavage rates of iSCNT embryos among the types of serum. The iSCNT embryos could not develop to the blastocyst stage, irrespective of the type of donor cell and serum. In conclusion, our results indicate that the bovine oocytes partly supported the remodelling and reprogramming of the canine somatic cell nuclei, but they were unable to support the development to the blastocyst stage of canine iSCNT embryos. Moreover, the development to the late embryonic stage of iSCNT embryos may be influenced by the type of donor cell but not serum.

Calreticulin expression in neoplastic versus normal dog mammary glands: A cDNA subtraction-based study

This is the first report describing the expression of canine calreticulin (cCRT) in canine mammary gland tumour (MGT). Using cDNA subtraction method, it is found that mRNAs of CRT, cathepsin A, ovostatin, and lactotransferrin were differentially expressed in mammary adenocarcinoma as compared to hyperplasia, both of which were obtained from the dog. Furthermore, the mRNA expression levels of CRT and cathepsin A were significantly higher in canine MGT samples than in nontumour samples. In contrast, immunohistochemical studies have indicated that the expression of cCRT protein found to be detected in most of mammary gland tissues and was not correlated to the types of canine MGTs. Furthermore, cCRT was molecularly cloned, and the amino acid sequence of cCRT was found to be very similar to those of other species. Further studies are required to elucidate additional roles of cCRT in canine MGT.

Epidermal growth factor enhances the malignant phenotype in canine mammary carcinoma cell lines

Canine mammary gland tumours (CMTs) are the most common malignancies in female dogs. The receptor tyrosine kinase EGFR (erbb1), a receptor for epidermal growth factor (EGF) and related factors, mediates multiple oncogenic functions in human epithelial neoplasms. While previous studies have demonstrated EGFR expression in canine tumours, its function has not been studied in canine cancer. The purpose of this study was to determine the in vitro effects of EGF and vandetanib (ZD6474), a small molecule inhibitor of VEGFR-2, EGFR and RET tyrosine kinases, on proliferation, invasion, survival and chemosensitivity in CMT cells. In low serum, EGF enhanced proliferation and chemotaxis, attenuated apoptosis, and stimulated vascular endothelial growth factor (VEGF) production. Vandetanib dose-dependently inhibited EGFR phosphorylation as well as PI3K/Akt activation, and inhibited all EGF-induced phenotypic effects. In conclusion, EGF stimulates multiple features promoting the malignant phenotype in CMT. Thus, CMT may be an important translational model for the investigation of novel EGFR-directed therapies.

Haemoglobin in normal and neoplastic canine mammary glands

Four types of globins for oxygen transport are known in vertebrates, and the haemoglobin is responsible for carrying oxygen in blood. In this study, we found that haemoglobin was also expressed in canine mammary glands. Samples were taken from 26 malignant mammary tumors, 16 normal mammary glands and 10 other normal tissues. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE), immunoblotting and mass spectrometry were used to investigate haemoglobin in mammary tissues. The results indicated that normal canine mammary glands expressed high levels of haemoglobin protein as shown by Coomassie blue staining. The identity of haemoglobin was confirmed by immunoblotting and mass spectrometry, and the mass spectrometry data revealed that both alpha-haemoglobin and beta-haemoglobin were expressed. Relative to normal mammary glands, the levels of haemoglobin expression in mammary tumors were lower. Our results also indicated that the haemoglobin was endogenously produced in mammary gland tissues and was not derived from the erythroid cells.

Insulin receptor is expressed in normal canine mammary gland and benign adenomas but decreased in metastatic canine mammary carcinomas similar to human breast cancer

Insulin receptor (INSR) or insulin-like growth factor (IGF) signalling is speculated to be involved in mammary tumour development. Expression levels of members of the insulin receptor family (INSR, IGF1R, IGF2R, GHR) and their ligands IGF1and IGF2 were quantified in macro- and microdissected tissue samples of normal canine mammary gland, adenomas, carcinomas and their lymph node metastases to evaluate their potential impact on the carcinogenesis of canine mammary tumours. Normal mammary gland and adenomas had strong INSR expression, while carcinomas and metastases had significantly decreased expression. No differences were observed for IGF1R expression. IGF1, IGF2 and GHR mRNA expressions were strongly decreased in adenomas, carcinomas and metastases. INSR and IGF1R are therefore expressed in normal gland and adenomas and an increased stimulus by their ligands may be a proliferative stimulus in those tissues. However, decreased INSR expression carcinomas and their metastases render questionable its impact at late stages of carcinogenesis.

Tryptase-Positive Mast Cells Correlate with Angiogenesis in Canine Mammary Carcinoma

Angiogenesis plays a crucial role in tumour growth, invasion and metastasis. Mast cells (MCs) release angiogenic factors that promote endothelial proliferation and differentiation. Previous studies have suggested that MCs are involved in tumour angiogenesis due to the release of various pro-angiogenic factors. This study evaluated samples from 40 canine mammary carcinomas and eight healthy non-neoplastic canine mammary glands. Toluidine blue staining was performed to characterize the MCs. Immunohistochemical labelling was performed to detect the number of tryptase-positive MCs and microvessels. MCs accumulated in tumour tissue and were closely associated with blood or lymphatic vessels in the tumour microenvironment. Angiogenesis, as measured by microvessel density, increased in direct proportion to the number of MCs. The correlation coefficient was significantly higher for tryptase-positive MCs than for toluidine blue-stained MCs. These results suggest that MCs are involved in tumour angiogenesis, which in turn influences tumour growth, invasion and metastasis. In particular, MC tryptase may be influential in mediating this function of MCs.

Immuno-histochemical staining expression of Ki67 protein in canine mammary gland carcinoma

Canine mammary gland carcinoma is ranked second after skin tumours in dogs in terms of frequency. Due to certain microscopic and biological features, canine tumours are very similar to human carcinomas, which may make the dog a good animal model. The objective of this study was to describe Ki67 protein expression properties using immuno-histochemical (IHC) staining using human antibody. In this study, 30 mammary gland tumour samples were studied. The histological diagnosis was made on the basis of the current World Health Organization classification for canine mammary tumours. Tumours were then graded histologically in accordance with the Elston and Ellis method for human breast tumours. IHC staining using MIB-1 antibody for detecting expression of Ki67 proteins (pKi67), was performed on canine mammary gland carcinoma tissue. Results from the semi-quantitative IHC assessment of pKi67 showed that 20% were negative, 20% were one plus, 36.5% were two plus, 23.3% were three plus. There was a significant difference between histological grade and pKi67 expression. This method would help the pathologist and clinician in determining a more accurate prognosis and treatment in canine mammary carcinoma and facilitate comparative studies of canine and human research.

Increased Presence of Stromal Myofibroblasts and Tenascin-C With Malignant Progression in Canine Mammary Tumors

The aims of this study were to determine whether the appearance of stromal myofibroblasts and the expression of tenascin-C (Tn-C) correlate with the grade of malignancy in canine mammary tumors and to determine the main cellular source of Tn-C in these tumors. Single or double immunostaining using antibodies against α-smooth muscle actin (α-SMA) and Tn-C was performed on serial sections of normal canine mammary glands as well as those with lobular hyperplasia, simple adenoma, and simple carcinoma. Thirty-nine of 42 simple carcinomas (93%) exhibited stromal α-SMA-positive myofibroblasts and Tn-C expression. Only 6 of 11 cases of simple adenoma (55%) showed these changes, whereas no changes were observed in normal mammary gland tissue or cases of lobular hyperplasia. The distribution of stromal Tn-C correlated with the presence of myofibroblasts. However, Tn-C immunoreactivity was also occasionally observed in the basement membrane zone surrounding the myoepithelial layer in normal tissue, benign lesions, and tubulopapillary carcinomas. This pattern of staining was not related to the presence of myofibroblasts. The appearance of stromal myofibroblasts and expression of Tn-C were significantly correlated with higher histological grades of malignancy and vascular/lymphatic invasion in simple carcinomas. Stromal myofibroblasts appear to be a major cellular source of Tn-C and play an important role in the development of canine mammary tumors. The Tn-C expressed in the basement membrane zone of normal, hyperplastic, and neoplastic mammary tissue, which is likely produced by neighboring myoepithelial cells, may differ functionally from the Tn-C produced by myofibroblasts.

744 WWOX tumour suppressor gene is involved in Wilms tumour cancerogenesis in a haploinsufficiency way

Canine mammary tumours (CMTs) are the most common tumours of entire female dogs and represent a promising model for human breast cancer. Little is known about the presence and prognostic value of lymphangiogenesis in CMTs. The aims of the present study were to analyze selected characteristics of lymphatic vessels in CMTs, to evaluate their prognostic significance and to compare these results with studies of human breast cancer. Fifty-six benign CMTs, 55 malignant CMTs and 13 control samples of normal canine mammary gland tissue were studied. Serial immunohistochemical labelling with the lymphatic marker prox-1 and the proliferation marker Ki67 was performed. In intratumoural (IT) and peritumoural (PT) regions, the lymphatic vessel density (LVD), mean lymphatic vessel perimeter (LVP) and relative area occupied by lymphatic vessels (LVA) were analyzed. Lymphatic endothelial cell proliferation (LECP) and tumour cell proliferation (TCP) were also measured. Lymphatic vessels were identified in IT and PT regions and lymphangiogenesis was present in both regions. The IT lymphatic vessels were smaller, less numerous and occupied a smaller relative area compared with those of the PT region. Although no differences in lymphatic vessel parameters were observed between benign and malignant tumours, control tissue differed significantly from neoplastic tissue. None of the lymphatic vessel parameters showed a prognostic value, except for LECP in PT regions of benign tumours. The findings were in accordance with results of investigations into human breast cancer, which supports the use of dogs with spontaneously occurring CMTs as an animal model in comparative oncology trials.

Expression of the plasma membrane markers aquaporin 1 (AQP1), glucose transporter 1 (GLUT1) and Na, K-ATPase in canine mammary glands and mammary tumours

This study investigated the expression of the plasma membrane markers aquaporin 1 (AQP1), glucose transporter 1 (GLUT1) and the alpha1 subunit of Na, K-ATPase in normal canine mammary glands and in benign and malignant mammary tumours, using immunohistochemistry and semi-quantitative histomorphometry. AQP1 immunoreactivity was absent from the majority of specimens studied. GLUT1 immunoreactivity was observed in normal mammary tissue and particularly in the epithelial and mesenchymal cells of benign, and in the epithelial cells of malignant tumours, respectively. Na, K-ATPase immunoreactivity was present in normal and neoplastic mammary epithelium and was significantly increased in the epithelium of both benign and malignant tumours. These results suggest that GLUT1 is more highly expressed in neoplastic epithelium and mesenchyme and that Na, K-ATPase is more highly expressed in neoplastic mammary epithelium. In consequence, these membrane proteins may have potential as diagnostic and prognostic biomarkers of canine mammary neoplasia.

Transcriptomic signature of cell lines isolated from canine mammary adenocarcinoma metastases to lungs

Metastasis is a final step in the progression of mammary gland cancer, usually leading to death. Potentially, a molecular signature of metastasis can be defined via comparison of primary tumors with their metastases. Currently, there is no data in the literature regarding the molecular portrait of metastases in dogs and only few reports regarding human cancer. This is the first report describing the transcriptomic signature of canine cancer metastatic cells. Two adenocarcinoma cell lines isolated from the canine mammary gland (CMT-W1 and CMT-W2) were compared with cell lines isolated from their lung metastases (CMT-W1M and CMT-W2M) with regards to the following cytometric parameters: cell cycle, ploidy, Bcl-2 expression, susceptibility to induced apoptosis, and transcriptomic profile. Cytometric analyses revealed significant differences in cell cycle and antiapoptotic potential between the examined cells. Using oligonucleotide microarrays, we found 104 up-regulated genes in the metastatic cell line CMT-W1M and 21 up-regulated genes in the primary CMT-W1 cell line. We also found 83 up-regulated genes in the CMT-W2M cell line and only 21 up-regulated genes in the CMT-W2 cell line. Among the up-regulated genes in both metastatic cell lines, we found 15 common genes. These differently expressed genes are involved mainly in signal transduction, cell structure and motility, nucleic acid metabolism, developmental processing, and apoptosis (GHSR, RASSF1, ARF1GAP, WDR74, SMOC2, SFRP4, DIAPH1, FSCN1, ALX4, SNX15, PLD2, WNT7B, POU6F2, NKG7, and POLR2F). Seven of them are involved in a cellular pathway dependent on ghrelin via growth hormone secretagogue receptor (GHSR). Our results suggest that this pathway may be essential for mammary cancer cells to have a metastatic potential.

Columnar cell lesions of the canine mammary gland: pathological features and immunophenotypic analysis

BackgroundIt has been suggested that columnar cell lesions indicate an alteration of the human mammary gland involved in the development of breast cancer. They have not previously been described in canine mammary gland. The aim of this paper is describe the morphologic spectrum of columnar cell lesions in canine mammary gland specimens and their association with other breast lesions.MethodsA total of 126 lesions were subjected to a comprehensive morphological review based upon the human breast classification system for columnar cell lesions. The presence of preinvasive (epithelial hyperplasia and in situ carcinoma) and invasive lesions was determined and immunophenotypic analysis (estrogen receptor (ER), progesterone receptor (PgR), high molecular weight cytokeratin (34βE-12), E-cadherin, Ki-67, HER-2 and P53) was perfomed.ResultsColumnar cell lesions were identified in 67 (53.1%) of the 126 canine mammary glands with intraepithelial alterations. They were observed in the terminal duct lobular units and characterized at dilated acini may be lined by several layers of columnar epithelial cells with elongated nuclei. Of the columnar cell lesions identified, 41 (61.2%) were without and 26 (38.8%) with atypia. Association with ductal hyperplasia was observed in 45/67 (67.1%). Sixty (89.5%) of the columnar cell lesions coexisted with neoplastic lesions (20 in situ carcinomas, 19 invasive carcinomas and 21 benign tumors). The columnar cells were ER, PgR and E-cadherin positive but negative for cytokeratin 34βE-12, HER-2 and P53. The proliferation rate as measured by Ki-67 appeared higher in the lesions analyzed than in normal TDLUs.ConclusionsColumnar cell lesions in canine mammary gland are pathologically and immunophenotypically similar to those in human breast. This may suggest that dogs are a suitable model for the comparative study of noninvasive breast lesions.

Abstract B58: Gene expression study on a highly aggressive canine transmissible venereal tumor of a NOD/SCID mice model

Abstract We have set up a xenograft NOD/SCID mice model bearing canine transmissible venereal tumor (CTVT) (designated as MCTVT). In addition to the similarity of the tumor cells between CTVT and MCTVT, the insertion of the specific gene fragment in the 5′ end of c-myc characteristic for a CTVT cell was also confirmed in MCTVT. However, as inoculating back to dogs, the MCTVT showed a much more aggressive growth and much higher metastatic frequency than a common CTVT. The goal of this research was to explore the gene expression differences between CTVT and MCTVT, and tried to delineate the mechanisms of their growth pattern differences. The differential gene expressions between the 2 tumors were studied using Affymetrix. Studied by hierarchical analysis and filtering the differential expressed genes by 2-fold method, we have found 105 genes significantly up-regulated and 37 down-regulated in MCTVT comparing to CTVT. Many of these genes were previously published to be involved in the areas such as malignant candidate genes, important biological processes, tumor development and immune modulation. We then used Real-Time RT-PCR to study the top 30 up-regulated genes. Three genes (APOC1, MMP1 and KMO) were further confirmed that were significantly up-regulated in MCTVT. The three genes were then tested in 22 spontaneous canine mammary gland tumors (CMGT) using Real-Time RT-PCR, the results indicated that two out of the three up-regulated genes discriminated malignant from benign CMGT and their gene expression was significantly higher in grades 4 and 5 malignant CMGT than those from lower grade tumors. We are currently studying the activities of KMO in association with the tumor cell growth. This study presented the novel candidate gene (KMO) that might associate with the malignant growth of tumor cells and has potential to be used as a biological marker to differentiate degrees of malignancy. Citation Information: Cancer Prev Res 2010;3(1 Suppl):B58.